Therapeutic proteins provide enormous potential for the treatment of human disease. Dozens of protein therapeutics are currently available, with hundreds more in clinical development (PhRMA 2004). Such proteins include human growth hormone, which is used to treat abnormal height when insufficient growth hormone is produced in the body, and interferon-gamma, which is used to treat neoplastic and viral diseases. Protein pharmaceuticals are often produced using recombinant DNA technology, which can enable production of higher amounts of protein than can be isolated from naturally-occurring sources, and which avoids contamination that often occurs with proteins isolated from naturally-occurring sources. Unfortunately, protein aggregation is a common problem that arises during all phases of recombinant protein production, specifically during fermentation, purification, and long term storage (Baneyx 1999; Carpenter et al. 1997; Chi et al. 2003; Clark 2001; Schwarz et al. 1996).
Proper folding of a protein is essential to the normal functioning of the protein. Improperly folded proteins are believed to contribute to the pathology of several diseases, including Alzheimer's disease, bovine spongiform encephalopathy (BSE, or “mad cow” disease) and human Creutzfeldt-Jakob disease (CJD), and Parkinson's disease; these diseases serve to illustrate the importance of proper protein folding.
Several proteins of therapeutic value in humans, such as recombinant human growth hormone and recombinant human interferon gamma, can be expressed in bacteria, yeast, and other microorganisms. While large amounts of proteins can be produced in such systems, the proteins are often misfolded, and often aggregate together in large clumps called inclusion bodies. The proteins cannot be used in the misfolded, aggregated state. Accordingly, methods of disaggregating and properly refolding such proteins have been the subject of much investigation.
One method of refolding proteins uses high pressure on solutions of proteins in order to disaggregate, unfold, and properly refold proteins. Such methods are described in U.S. Pat. Nos. 6,489,450, 7,064,192, U.S. Patent Application Publication No. 2004/0038333, and International Patent Application WO 02/062827. Those disclosures indicated that certain high-pressure treatments of aggregated proteins or misfolded proteins resulting in recovery of disaggregated protein retaining biological activity (i.e., the protein was properly folded, as is required for biological activity) in good yields. U.S. Pat. No. 6,489,450, U.S. 2004/0038333, and WO 02/062827 are incorporated by reference herein in their entireties. Additional studies (see U.S. Pat. App. Pub. No. 2005/0020818, Randolph et al. 2002; Seefeldt et al. 2004; St. John et al. 1999; St. John et al. 2002) have also shown that high hydrostatic pressure (c. a. 2000 bar) can be an effective refolding tool, enabling refolding at relatively high concentration and with high yield. In contrast to traditional chaotrope-based refolding, pressures can be selected that dissociate aggregates under conditions that favor the protein's native conformation (Seefeldt et al. 2004; St. John et al. 2002). High pressure refolding has been particularly effective when the aggregates contain non-native disulfide bonds, providing sufficient time for disulfide bond formation while inhibiting off-pathway aggregation reactions (Seefeldt et al. 2004; St. John et al. 2002).
High hydrostatic pressures (up to 4000 bar) have been shown to increase the catalytic activity of twelve enzymes and proteases (Hendrickx et al. 1998; Mozhaev et al. 1996). Although the mechanism remains unclear, it appears the high hydrostatic pressures increase reaction kinetics through enzymatic reactions that have negative activation volumes, while favoring or even stabilizing the native enzyme conformation (Mozhaev et al. 1996). Some proteases have also been found to be unusually stable at elevated pressures, which may be due to their low partial specific volume (Gekko 2002; Gekko and Hasegawa 1986; Seefeldt et al. 2003). These studies conclude that high hydrostatic pressures (c.a. 2000) maintain or, in some cases, increase activity of certain enzymes, although there is an upper limit and higher pressures can also inactivate enzymes through denationuration (Hendrickx et al. 1998; Mozhaev et al. 1996; Royer 2002).
For some proteins, prokaryotic expression in E. coli. is further complicated by the need for co-expression with fusion partners as a so-called fusion protein. Fusion proteins are often required to minimize the lethality of expressed proteins or improve solubility (Baneyx 1999; Sorensen and Mortensen 2005). For example, therapeutic recombinant human insulin and recombinant human insulin-like growth factor can also modulate bacterial cellular processes. These properties necessitate the use of fusion proteins and co-expression for industrial scale production. Fusion proteins facilitate prokaryotic expression, however they must be removed for final drug production (Baneyx 1999; Sorensen and Mortensen 2005).
Currently, the main disadvantages of fusion-protein technologies are: First, liberation of the passenger proteins requires expensive protease (e.g. Factor Xa and enterokinase). Secondly, cleavage is rarely complete, leading to reduction in yields. Thirdly, additional processing steps may be required to obtain an active product (Baneyx 1999; Sorensen and Mortensen 2005). Traditionally, chaotropes are required to facilitate solubilization, but also denature native proteins, decreasing protease stability and effectiveness (Timasheff 1993).
Thus there is a need for improved processes for efficiently and cost-effectively solubilizing and cleaving fusion protein aggregates to produce useful recombinant proteins for use in therapeutic and industrial settings. The methods provided herein use high hydrostatic pressure to solubilize fusion protein aggregates produced during prokaryotic expression and cleave the fusion protein efficiently with minimal manipulation of the fusion protein aggregates and resulting protein of interest. If protease cleavage can occur in the presence of disulfide shuffling agents or the protein of interest does not contain disulfide bonds, refolding can also occur during solubilization and cleavage process, thus providing a highly efficient and cost-effective single step method for solubilization/cleavage/refolding.